Nanostructured Thin Films and Their Uses

ABSTRACT

The present invention generally discloses the use of a nanostructured non-silicon thin film (such as an alumina or aluminum thin film) on a supporting substrate which is subsequently coated with an active layer of a material such as silicon or tungsten. The base, underlying non-silicon material generates enhanced surface area while the active layer assists in incorporating and transferring energy to one or more analytes adsorbed on the active layer when irradiated with a laser during laser desorption of the analyte(s). The present invention provides substrate surfaces that can be produced by relatively straightforward and inexpensive manufacturing processes and which can be used for a variety of applications such as mass spectrometry, hydrophobic or hydrophilic coatings, medical device applications, electronics, catalysis, protection, data storage, optics, and sensors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/226,075, filed Sep. 14, 2005, which application claims the benefit ofU.S. Provisional Patent Application No. 60/611,116, filed Sep. 17, 2004,which is incorporated in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Portions of this invention may have been made with United StatesGovernment support under the Department of Health and Human Services,National Institutes of Health, National Human Genome Research Institutegrant number 1 R43 HG003480-01. As such, the United States Governmentmay have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates primarily to the field of nanotechnology.More specifically, the invention pertains to nanostructured thin filmsand coatings and their uses in, e.g., high surface area applicationssuch as mass spectrometry, as super-hydrophobic coatings, asanti-bifouling coatings, as surfaces to promote cell attachment,differentiation and proliferation, etc.

BACKGROUND OF THE INVENTION

Nanotechnology has been heralded as the next major technological leap,in that it is prophesied to yield a variety of substantial advantages interms of material characteristics, including electronic, optical andstructural characteristics. Nanostructured materials as thin films andcoatings possess unique properties due to both size and interfaceeffects. Nanostructured materials are generally a broad class ofmaterials, with microstructures modulated in zero to three dimensions onlength scales typically less than about 500 nm, for example, less thanabout 200 nm, e.g., less than about 100 nm. Nanostructured materialsfind many applications in areas such as electronics, mass spectrometry,catalysis, protection, data storage, optics, and sensors. Nanostructuredfilms and coatings have many advantages over conventional thin filmsincluding high surface area, increased hydrophobicity, increasedadhesion, and other similar properties.

One particularly interesting use of nanostructured films and coatings isin mass spectrometry applications. Generally speaking, in massspectrometry, a substance is bombarded with an electron beam havingsufficient energy to fragment the molecule. The positive fragments whichare produced (cations and radical cations) are accelerated in a vacuumthrough a magnetic field and are sorted on the basis of mass-to-charge(m/z) ratio in a mass analyzer. Since the bulk of the ions produced inthe mass spectrometer carry a unit positive charge, the value m/z isequivalent to the molecular weight of the fragment. Modern advances inmass spectrometry often address problems regarding the handling ofliquid or solid samples. As ions are actually analyzed in the vacuum ofthe mass spectrometer, arguably the most important reaction is the onethat converts analytes of interest into gas-phase ions. Historically,the most commonly used ionization processes (for example, electronionization) occur in two discrete steps: a sample which is adsorbed on asurface of a substrate is first volatilized and then ionized.

The past two decades have seen the development of new ionizationtechniques for the analysis of non-volatile and thermally labilecompounds: Electrospray ionization (ESI) and matrix-assisteddesorption/ionization (MALDI). ESI allows for large, non-volatilemolecules to be analyzed directly from the liquid phase. Rather thanusing an electron beam to ionize a sample as with ESI, MALDI ionizes asample by pulsed laser irradiation of the sample. The sample isco-crystallized with a solid matrix that can absorb the wavelength oflight emitted by the laser. Usually the sample and matrix are mixed on asubstrate and inserted into the mass spectrometer instrument, and afterirradiation the gas-phase ions that are formed are directed toward themass analyzer. The broad success of matrix-assisted laserdesorption/ionization (MALDI) is related to the ability of the matrix toincorporate and transfer energy to the sample. Barber, et al., Nature293, 270-275 (1981); Karas, et al., Anal. Chem. 60, 2299-2301 (1988);Macfarlane, et al., Science 191, 920-925 (1976); Hillenkamp, et al.,Anal. Chem. 63, A1193-A1202 (1991)).

However, one of the drawbacks of MALDI is the presence of the matrix,which facilitates ionization, but also causes a large degree of chemicalnoise to be observed at m/z ratios below about 700 Daltons (e.g., forlow molecular weight samples). As a result, samples with low molecularweights are usually difficult to analyze with MALDI. Recent variationsof MALDI have involved direct desorption/ionization without a matrix andhave potential for enabling the analyses of low molecular weightcompounds. In particular, the desorption/ionization on porous silicon(DIOS) and silicon continuous or columnar thin films has been used as analternative to MALDI, see, e.g., Siuzdak et al. U.S. Pat. No. 6,288,390;Fonash et al. U.S. Patent Application No. 20020048531 filed Dec. 19,2000; Thomas, J. J., Shen, Z., Crowell, J. E., Finn, M. G. & Siuzdak,G., “Desporption/ionization on silicon (DIOS): a diverse massspectrometry platform for protein characterization,” Proc. Natl Acad.98:4932-4937 (2001); Shen, Z., et al., “Porous silicon as a versatileplatform for laser desorption/ionization mass spectrometry,” Anal Chem.73:612-619 (2001); Cuiffi, et al., “Desorption-ionization massspectrometry using deposited nanostructured silicon films,” Anal, Chem.73:1292-1295 (2001); and, Kruse, et al., “Experimental factorscontrolling analyte ion generation in laser desorption/ionization massspectrometry on porous silicon,” Anal. Chem. 73:3639-3645 (2001). Thesemethods typically use porous silicon or etched silicon columnarstructures to trap analytes deposited on the surface, and laserirradiation to vaporize and ionize them. Most of these demonstratedapplications to date have been based on the porous silicon materialproduced by electrochemically etching a wafer or deposited film ofsilicon.

Silicon nanowires have been the subject of extensive research inelectronics, photonics, optoelectronics, sensing, and other novel deviceapplications. See, e.g., Cui, et al., “Nanowire nanosensors for highlysensitive and selective detection of biological and chemical species,”Science 293;1289-1292 (2001); Cui, et al., “Functional nanoscaleelectronic devices assembled using silicon nanowire building blocks,”Science 291:851-853 (2001); Huang, et al., “Integrated optoelectronicsassembled from semiconductor nanowires,” Abstracts of Papers of theAmerican Chemical Society 224:U308 (2002); Zhou, et al., “Siliconnanowires as chemical sensors,” Chem. Phys. Lett. 369:220-224 (2003);Duan, et al., “Single-nanowire electrically driven lasers,” Nature421:241-245 (2003); Hahm, et al., “Direct ultrasensitive electricaldetection of DNA and DNA sequence variations using nanowirenanosensors,” Nano Lett. 4:51-54 (2004). Silicon nanowires appear to bean ideal platform for surface-based mass spectrometry. In contrast toporous silicon, silicon nanowires are catalyzed and grown on the surfaceof a substrate and their physical dimensions, composition, density, andposition can be precisely controlled at the nanoscale level, thusoffering even greater potential for designing mass spectrometry activesurfaces. See, e.g., U.S. Ser. No. 60/468,390 filed May 6, 2003, U.S.Ser. No. 60/468,606 filed May 5, 2003, and U.S. Ser. No. 10/792,402filed Mar. 2, 2004, all three entitled “Nanofiber Surfaces for Use inEnhanced Surface Area Applications”, the entire contents of which areincorporated by reference herein. However, the use of silicon nanowiresas substrate surfaces may pose some challenges in terms of manufacturingsuch surfaces reproducibly for large-scale commercial production.

It would be beneficial to have a direct laser desorption/ionizationtechnique that eliminates the need for matrix compounds, is reliable andrelatively inexpensive to implement, and can be used in biomolecular andother analyses with standard MALDI (and other) mass spectrometerinstruments. The present invention provides unique nanostructured thinfilm surfaces to generate high surface area substrates for matrix-freeMALDI and other applications as well. By eliminating background peaks ofinterfering matrix compounds, good analyses of both low andhigh-molecular weight compounds such as small molecules, proteins,peptides, oligonucleotides, drugs, pesticides, carbohydrates, fattyacids and the like can be produced more quickly and reliably.

SUMMARY OF THE INVENTION

The present invention generally discloses the use of a nanostructurednon-silicon thin film (such as an alumina or aluminum thin film) on asupporting substrate which is subsequently coated with an active layerof a second material (e.g., silicon, tungsten, etc.). The baseunderlying non-silicon material generates enhanced surface area whilethe active layer assists in incorporating and transferring energy to oneor more analytes adsorbed on the active layer when irradiated with alaser during laser desorption of the analyte(s). The present inventionprovides substrate surfaces that can be produced by relativelystraightforward and inexpensive manufacturing processes. Thenanopatterned thin film produced by the methods of the present inventionhas characteristics of high uniformity over a relatively large area,strong adhesion to the underlying substrate, resistance to scratching,and can be easily applied onto large substrate materials. Thenanostructured film may be used as a base layer for increasing thesurface area of a substrate, e.g., for matrix-free mass spectrometryapplications, for making a substrate surface superhydrophilic orsuperhydrophobic, for modifying the optical properties of a substrate,and/or for developing highly efficient catalytic surfaces, e.g., for thegrowth of nanofibers or other nanostructured components thereon.

In a first exemplary aspect of the invention, a device is disclosedwhich generally comprises a supporting substrate, a first layer of ananostructured coating, and a second layer of an active coating. Thesupporting substrate can take a variety of forms such as a semiconductorwafer, a sheet of glass or quartz, a sheet of metal, a piece of ceramicor plastic. The first layer of nanostructured coating can comprise, forexample, elongated or plate-like grains with at least one dimension lessthan about 0.2 μm. The long axis of plate-like grains may be orientedsubstantially perpendicular to a surface of the supporting substrate. Inone aspect of the invention, the first layer of nanostructured coatingcomprises an insulating inorganic material such as a native oxide layeror a deposited oxide or nitride layer. The insulating inorganic materialmay also be formed form a deposited metal layer. For example, theinsulating inorganic material may be selected from the group ofmaterials including alumina (Al₂O₃), aluminum (Al), SiO₂, ZrO₂, HfO₂, ahydrous form of these oxides, a compound oxide such as SiTiO₃, BaTiO₃PbZrO₄ or a silicate.

In one aspect of the invention, the active coating layer comprises asemiconductor which absorbs light energy such as IR, red, green, blueand/or UV laser light energy. The semiconductor may be selected fromGroup IV semiconductors including Si, Ge, diamonds and diamond-likecarbon coatings, or compound semiconductors of Group II-V, II-VI andother semiconductors including sulfides, selenides, tellurides,nitrides, carbides, nitrides, antimonides, and phosphides. The activelayer may also comprise a metal such as tungsten or other metal which iscatalytically active and interferes with light absorption, and which iscapable of promoting or enhancing light energy transfer to an analyte,e.g., to ionize and desorb it from the surface of the supportingsubstrate. The metal may be selected from the group comprisingtransition metals including Fe, Co, Cr, Ni, Mo, W, V, Cu, Zn and precisemetals including Ag, Au, Pt, Pd, Ru, Rh, or metal alloys thereof.

In another aspect of the invention, a mass spectrometry device isdisclosed which comprises a supporting substrate having a first surfaceand a thin non-silicon film layer of a first material deposited on atleast a region of the first surface, the film layer having ananostructured surface; an active layer of a second material depositedon the first layer; and at least a first analyte positioned in contactwith at least a region of the active layer, wherein the active layerassists in incorporating and transferring energy to the at least firstanalyte when irradiated with a laser during laser desorption of theanalyte. The non-silicon film layer can be deposited on the substrateusing a variety of well-known techniques such as thermal evaporation andsputtering including physical vapor deposition (PVD), sputterdeposition, low or high temperature chemical vapor deposition (CVD),metallorganic CVD, plasma-enhanced CVD, laser ablation, or usingsolution deposition methods such as spray coating, dip coating, or spincoating etc. Ultra-thin metal films (e.g., films less than about 5 nm inthickness) may be deposited by atomic layer deposition (ALD) techniques.The thin film may be configured to have a nanostructured surface by, forexample, simply exposing the film to hot water or water vapor. For thispurpose, the thin film surface is exposed to hot water or water vapor ata temperature greater than about 50 degrees Celsius, e.g., between about50 to 150 degrees Celsius. The water or the water vapor preferably has atemperature of from about 90 to 125 degrees Celsius. The film surface isexposed to the water or water vapor for a sufficient time (e.g., betweenabout 3 to 60 minutes, for example, between about 5 and 30 minutes) toconvert the film into a highly ordered nanostructured surface. In oneembodiment, the thin film is a metal thin film made from alumina oraluminum. Other textured surfaces can also be used as long as suchsurfaces enhance desorption sensitivity, including, for example, anative oxide layer or a deposited oxide or nitride layer or materialsincluding ZnO, SiO₂, ZrO₂, HfO₂, a hydrous form of these oxides, acompound oxide such as SiTiO₃, BaTiO₃ PbZrO₄ or a silicate. In otherembodiments, the supporting substrate itself is made from a non-siliconmaterial such as alumina or aluminum from which a nanostructured surfacecan be formed, and the active layer is deposited on the nanostructuredsurface of the substrate.

The active layer may comprise a semiconductor material such as siliconor germanium, or other conducting or semiconducting materials, such asmetals and semimetals, and other materials which absorb light andpromote or enhance light energy transfer to an analyte to ionize anddesorb it from the surface of the supporting substrate as describedabove and further below. In one example, the active layer is made fromsilicon, and the silicon active layer is deposited on the thin filmlayer by a chemical vapor deposition process for ten or more minutes toa depth of about 50 nm or more, or by other thermal evaporation methodsuch as sputtering. The active layer can also optionally be coated orfunctionalized, e.g., to enhance or add specific properties. Forexample, polymers, ceramics, or small molecules can optionally be usedas coating materials. The optional coatings can impart characteristicssuch as water resistance, improved mechanical, optical (e.g.,enhancement of light coupling) or electrical properties or specificitiesfor certain analytes. The active layer may also be derivatized with oneor more functional moieties to enhance laser desorption such as one ormore silane groups, e.g., one or more per-fluorinated silane groups, orother coatings such as diamond-like carbon thin film coatings (e.g., torender the surface of the film hydrophobic).

The supporting substrate may be made from any one of a number ofconventional materials including stainless steel, glass, quartz,semiconductor materials such as silicon, polymers, ceramics etc. Samplesof the substances (e.g., small molecules, proteins, peptides etc.) to beanalyzed are optionally placed in contact with the active layer (e.g.,directly or via the use or one or more functional moieties such as oneor more silane groups) by conventional dispensing means such aspipetting, dot-printing etc. Those of skill in the art will be familiarwith various protocols to follow to dry the samples for analysis. Laserenergy levels and pulse durations are also optionally optimized foranalysis of the samples arrayed upon the nanostructured substratesurface. Again, those of skill in the art will be familiar with ways ofdetermining optimal parameters for laser energy, pulse time, etc. formass spectrometry. For example, the different parameters are optionallymodified depending upon, e.g., the specific molecules being detected.For example, the laser energy used can optionally be adjusted (e.g.,higher laser energy levels for peptides as opposed to small molecules).

In another embodiment of the invention, a mass spectrometry system isdisclosed which generally comprises a substrate comprising a firstsurface and a thin non-silicon film layer of a first material (e.g., ametal such as alumina or aluminum) on at least a region of the firstsurface, the film having a nanostructured surface; an active layer of asecond material deposited on the first layer; at least a first analytepositioned in contact with a first region of the active layer; a laserpositioned to direct energy at the at least first region to desorb theat least first analyte from the first region; and a mass spectrometerinstrument positioned to receive the at least first analyte desorbedfrom the active layer, wherein the active layer assists in incorporatingand transferring energy to the at least first analyte during laserdesorption. The active layer may be functionalized or coated with one ormore functional moieties to enhance or add specific properties, e.g., torender its surface hydrophobic.

In other aspects of the present invention, methods are provided formanufacturing substrates for various applications including massspectrometry, as well as methods for detecting one or more analytes,e.g., for mass spectrometry analysis, using such substrates. In oneembodiment, a method for manufacturing a substrate device is disclosedwhich comprises providing a supporting substrate having a first surface;depositing a non-silicon film layer on the first surface; forming ananostructured surface from the non-silicon film layer; depositing anactive layer on the nanostructured surface; and depositing one or moreanalytes onto one or more regions of the active layer. The step ofdepositing a non-silicon film layer on the first surface of thesubstrate can comprise, for example, sputtering the film onto the firstsurface or using a CVD method to deposit the film on the surface of thesubstrate. The non-silicon film can comprise, for example, an alumina oraluminum film from which a nanostructured film surface may be easilyformed, or other similar nanostructured surface. The step of forming thenanostructured surface from the film layer can comprise, for example,exposing the film layer to hot water or water vapor at a temperature ofgreater than about 50 degrees Celsius, e.g., between about 50 to 150degrees Celsius, for example, between about 90 to 125 degrees Celsius,for between about three to sixty minutes, to form a highly structuredsurface. The step of depositing the active layer on the non-silicon filmlayer can comprise, for example, using a chemical vapor depositionprocess to deposit such film, wherein such film comprises a silicon ortungsten film in exemplary embodiments. In this way, substrates usefulfor a variety of applications such as mass spectrometry can bereproducibly produced easily and with relatively little cost, e.g., ascompared to wet electrochemically etched porous silicon films. Themethod may further comprise derivatizing the active layer with one ormore functional moieties to, e.g., increase the hydrophobicity of thesurface and/or to assist in desorbing the one or more analytes from thesurface of the active layer.

In a related aspect of the invention, a method for analyzing one or moreanalytes is provided which generally comprises providing a supportingsubstrate having a first surface and a thin non-silicon film layerdeposited on the first surface and an active layer deposited on the thinfilm layer, the thin film layer having a nanostructured surface; andanalyzing one or more analytes associated with the active layer by oneor more detection means such as mass spectrometry analysis. Inparticular, a method for performing mass spectrometry is disclosed whichcomprises providing a substrate having a first surface and a thin filmlayer (e.g., a non-silicon layer such as alumina or aluminum) depositedon the first surface and an active layer deposited on the thin filmlayer, the thin film layer having a nanostructured surface and one ormore analytes associated with the surface; desorbing the one or moreanalytes from the surface with energy from a laser directed at thesurface; and analyzing the one or more analytes in a mass spectrometerinstrument. The analytes to be measured include, for example, proteins,peptides, carbohydrates, fatty acids, small molecules, nucleic acids,cells, polypeptides and mixtures thereof. The analytes may be directlyapplied to the surface of the substrate in solid, dried form or inliquid form and dried on the substrate. The analytes may be suspended inaqueous or organic solutions or suspensions.

In another aspect of the present invention, a mass spectrometry deviceis disclosed which comprises a supporting substrate having a firstsurface and a plurality of nanocrystals, e.g., semiconductornanocrystals such as quantum dots, deposited on the surface of thesubstrate. For example, the nanocrystals can be a gold nanoparticle, acobalt nanoparticle, an iron oxide nanoparticle, or a semiconductornanocrystal including a semiconductor material, such as a Group IVcompound (e.g., Si, Ge), a Group II-VI compound, a Group II-V compound,a Group III-VI compound, a Group III-V compound, a Group IV-VI compound,a Group I-III-VI compound, a Group II-IV-VI compound, or a Group II-IV-Vcompound. The semiconductor material can be, for example, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, AlN, AIP, AlAs, AlSb, GaN, GaP,GaAs, GaSb, GaSe, InN, InP, InAs, InSb, TIN, TIP, TIAs, TlSb, PbS, PbSe,PbTe, or mixtures thereof. The nanoparticle can be coated with one ormore second materials such as a second metal, semimetal, polymer, orsemiconductor material such as ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe,CdTe, MgO, MgS, MgSe, MgTe, HgO, HgS, HgSe, HgTe, AIN, AIP, AlAs, AlSb,GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, TIN, TIP, TIAs, TlSb, TlSb,PbS, PbSe, PbTe, or mixtures thereof.

In another aspect of the invention, a device for enhanced surface areaand/or hydrophobic surface applications other than mass spectrometry(e.g., such as for use in medical device applications, buildingmaterials, barrier layers for storage tanks, a surface for cellattachment, differentiation and proliferation, contact touch screens foruse with ATM machines, in car displays, in entertainment portals, etc.)is disclosed which generally comprises a supporting substrate having asurface, a non-silicon film layer on at least a region of the surface,the film layer having a nanostructured surface, and a hydrophobic (orhydrophilic) coating deposited on the film layer. The hydrophobiccoating may comprise, for example, a diamond-like carbon coating orother coating which renders the surface of the substrate hydrophobic (orhydrophilic). The diamond-like carbon film can be applied to thenanostructured surface by, e.g., using pulsed laser ablation on agraphite target or a plasma assisted CVD method at low temperature. Whenthe deposited material is heated, such films lose their stress, yetretain their diamond-like properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a configuration of an exemplary laser desorption/ionizationmass spectrometry setup.

FIG. 2A is a cross-sectional view of a mass spectrometry substrateaccording to the teachings of the present invention.

FIG. 2B is an illustration of the substrate of FIG. 2A derivatized withfunctionalities by silylation.

FIG. 3 shows an SEM close-up image of a nanostructured alumina film on astainless steel substrate.

FIG. 4A shows an electron micrograph of a substrate having ananostructured alumina surface with an active silicon layer depositedthereon.

FIG. 4B is a cross-sectional electron micrograph view of the substratesurface of FIG. 4A.

FIG. 5A shows results of mass spectrometry analysis of samples on astainless steel substrate without any nanostructured thin film layer.

FIG. 5B shows results of mass spectrometry analysis of samples on astainless steel substrate with a silicon layer and without anyunderlying nanostructured thin film layer.

FIG. 5C shows results of mass spectrometry analysis of samples on astainless steel substrate with a thin nanostructured film layer and asilicon active layer according to the teachings of the present invention

FIG. 6A shows results of mass spectrometry analysis of 25 pg ofclonidine deposited on a silicon coated nanostructured alumina surface.

FIG. 6B shows results of mass spectrometry analysis of 25 pg ofhaloperidol deposited on a silicon coated nanostructured aluminasurface.

FIG. 6C shows results of mass spectrometry analysis of 25 pg of prazosindeposited on a silicon coated nanostructured alumina surface.

FIG. 6D shows results of mass spectrometry analysis of 25 pg ofquinidine deposited on a silicon coated nanostructured alumina surface.

FIG. 7A shows results of mass spectrometry analysis of 2.5 pg ofclonidine deposited on a silicon coated nanostructured alumina surface.

FIG. 7B shows results of mass spectrometry analysis of 2.5 pg ofhaloperidol deposited on a silicon coated nanostructured aluminasurface.

FIG. 7C shows results of mass spectrometry analysis of 2.5 pg ofprazosin deposited on a silicon coated nanostructured alumina surface.

FIG. 7D shows results of mass spectrometry analysis of 2.5 pg ofquinidine deposited on a silicon coated nanostructured alumina surface.

FIG. 8A shows results of mass spectrometry analysis of 25 pg ofclonidine deposited on a silicon coated nanostructured aluminumsubstrate surface.

FIG. 8B shows results of mass spectrometry analysis of 25 pg ofhaloperidol deposited on a silicon coated nanostructured aluminumsubstrate surface.

FIG. 8C shows results of mass spectrometry analysis of 25 pg of prazosindeposited on a silicon coated nanostructured aluminum substrate surface.

FIG. 8D shows results of mass spectrometry analysis of 25 pg ofquinidine deposited on a silicon coated nanostructured aluminumsubstrate surface.

FIG. 9A shows results of mass spectrometry analysis of 25 pg ofhaloperidol deposited on a silicon coated nanostructured alumina surfacethat had the silicon active layer deposited by CVD for a deposition timeof about 30 minutes.

FIG. 9B shows results of mass spectrometry analysis of 25 pg ofhaloperidol deposited on a silicon coated nanostructured alumina surfacethat had the silicon active layer deposited by CVD for a deposition timeof about 10 minutes.

FIG. 10A shows results of mass spectrometry analysis of 25 pg ofprazosin deposited on a tungsten coated nanostructured aluminumsubstrate surface.

FIG. 10B shows results of mass spectrometry analysis of 25 pg ofhaloperidol deposited on a tungsten coated nanostructured aluminumsubstrate surface.

FIG. 10C shows results of mass spectrometry analysis of 25 pg ofquinidine deposited on a tungsten coated nanostructured aluminumsubstrate surface.

FIG. 10D shows results of mass spectrometry analysis of 25 pg ofclonidine deposited on a tungsten coated nanostructured aluminumsubstrate surface.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes apparatus and methods of use of ananostructured non-silicon thin film (such as an alumina or aluminumthin film) on a supporting substrate which is subsequently coated withan active material layer. The base non-silicon material generatesenhanced surface area and the appropriate structural dimensions whilethe active layer assists in incorporating and transferring energy to oneor more analytes adsorbed on the active layer when irradiated with alaser during laser desorption of the analyte(s). In the followingdescription numerous specific details are set forth in order to providea thorough understanding of the present invention. One skilled in theart will appreciate that these specific details are not necessary inorder to practice the present invention. In other instances, well knownequipment features and processes have not been set forth in detail inorder to avoid obscuring the present invention.

FIG. 1 depicts a schematic configuration of an exemplary laserdesorption/ionization mass spectrometry set-up showing a MALDI plate 8customarily used in MALDI studies holding modified supporting substrate10 illuminated by a series of laser pulses 14. The supporting substrate10 can have a plurality of analytes 12 (e.g., proteins, peptides, smallmolecules etc.) adsorbed to it, preferably without the use of a matrixmaterial used in conventional MALDI analyses. The modified surface ofthe substrate absorbs the laser pulses 14 and ionizes and releases theanalytes to form a desorbed and ionized analytes 13. The desorbed andionized analytes 13 then travel to a mass spectrometry instrument (notshown) for analysis.

FIG. 2A shows an enlarged cross-sectional view of the modifiedsupporting substrate 10 according to the teachings of the presentinvention. The supporting substrate can take a variety of forms such asa semiconductor wafer, a sheet of glass or quartz, a sheet of metal, apiece of ceramic or plastic, and the like. As shown, the supportingsubstrate 10 has a first surface 20 and a thin non-silicon film layer 22of a first material deposited on at least a region of the first surface,the film layer having a nanostructured surface. An active layer 24 of asecond material is deposited on the first layer. A plurality of analytes12 are positioned in contact with at least a region of the active layer22, either directly or via the use of one or more functional groups asshown in FIG. 2B. Without being bound to any particular theory ofoperation, it is believed that the active layer assists in incorporatingand transferring energy (e.g., light energy from the laser pulses) tothe analytes when irradiated with a laser during laser desorption of theanalyte. For example, it is believed that the analyte molecules areionized by acid-base proton transfer reactions with the protonatedactive layer ions in a dense phase just above the surface of the activelayer. The protonated active layer molecules are generated by a seriesof photochemical reactions.

In one aspect of the invention, the first non-silicon layer may comprisean insulating inorganic material such as a native oxide layer or adeposited oxide or nitride layer. The insulating inorganic material mayalso be formed form a deposited metal layer. For example, the insulatinginorganic material may be selected from the group of materials includingaluminum (Al), alumina (Al₂O₃), ZnO, SiO₂, ZrO₂, HfO₂, a hydrous form ofthese oxides, a compound oxide such as SiTiO₃, BaTiO₃ PbZrO₄ or asilicate. In one example, the non-silicon film layer is made fromalumina or aluminum which can be deposited on the substrate using avariety of well-known techniques such as thermal evaporation andsputtering including physical vapor deposition (PVD), sputterdeposition, chemical vapor deposition (CVD), metallorganic CVD,plasma-enhanced CVD, laser ablation, or solution deposition methods suchas spray coating, dip coating, or spin coating etc. Ultra-thin metalfilms (e.g., films less than about 5 nm in thickness) may be depositedby atomic layer deposition (ALD) techniques. The thin film preferablyhas a thickness less than about 1000 nm, for example, between about 5and 400 nm, for example, between about 5 and 200 nm, for example,between about 10 and 100 nm. When an aluminum substrate is used, thethickness of the substrate may be significantly greater, e.g., on theorder of about 0.5 mm or thicker.

The inventors of the instant claimed invention have surprisinglydiscovered that the film layer may be configured to have ananostructured surface as shown, for example, in the SEM images of FIGS.3 and 4, by, for example, simply exposing the film to hot water or watervapor. For this purpose, the surface is exposed to hot water or watervapor at a temperature greater than about 50 degrees Celsius, e.g.,between about 50 to 150 degrees Celsius. The water or the water vaporpreferably has a temperature of from about 90 to 125 degrees Celsius.The surface is likewise preferably exposed to the water or water vaporfor a sufficient time (e.g., between about 3 to 60 minutes, for example,between about 5 to 30 minutes) to convert the film into a highly orderednanostructured surface having pore sizes less than about 200 nm. Forexample, FIGS. 4A-B are electron micrographs of a nanostructured aluminasurface with an active silicon layer deposited thereon. As shown, thenanostructured alumina layer comprises elongated or plate-like grainswith at least one dimension less than about 0.2 μm. The long axis of theplate-like grains are oriented substantially perpendicular to thesurface of the supporting substrate.

The nanostructured alumina layer can also be formed by other means suchas the formation of porous alumina films via the anodization of aluminummetal in acidic solution (e.g., phosphoric, oxalic, or sulfuric acidsolutions), autoclaving the film layer e.g., by placing the substrate ina commercially available gravity or vacuum autoclave device, and thelike. See, e.g., Evelina Palibroda, A. Lupsan, Stela Pruneanu, M. Savos,Thin Solid Films, 256, 101 (1995), the entire contents of which areincorporated by reference herein. Other textured surfaces other thanalumina or aluminum can also be used as long as such surfaces enhancedesorption sensitivity, including, for example, zinc oxide (ZnO)nanostructured surfaces described above. Low-temperature solution-basedapproaches to forming ZnO nanotextured surfaces are described, forexample, in “Low Temperature Wafer-Scale Production of ZnO NanowireArrays,” Lori E. Greene et al., Angew. Chem. Int. Ed. 2003, 42,3031-3034, the entire contents of which are incorporated by referenceherein.

The supporting substrate 10 may be made from a non-silicon material suchas aluminum from which a nanostructured surface can be formed (e.g., byheating the material with water or water vapor at a temperature greaterthan about 50 degrees Celsius, e.g., between about 50 to 150 degreesCelsius, e.g., at a temperature of from about 90 to 125 degrees Celsius,for a period of time sufficient to form the nanostructured surface,e.g., between about 3 and 60 minutes, e.g., preferably greater thanabout 5 minutes), and the active layer is deposited on thenanostructured surface of the substrate. For example, FIGS. 8A-D showmass spectrometry analysis of 25 picograms of four pharmaceuticalcompounds (e.g., clonidine, haloperidol, prazosin, and quinidine,respectively) deposited on a silicon coated nanostructured aluminumsubstrate surface.

The active layer 24 on top of the nanostructured film layer may comprisea semiconductor which absorbs light energy such as IR, red, green, blueand/or UV laser light energy. The semiconductor may be selected fromGroup IV semiconductors including Si, Ge, diamonds and diamond-likecarbon coatings, or compound semiconductors of Group II-V, II-VI andother semiconductors including sulfides, selenides, tellurides,nitrides, carbides, nitrides, antimonides, and phosphides. The activelayer may also comprise a metal such as tungsten (W) or other metalwhich is catalytically active and that interferes with light absorption,and which is capable of promoting or enhancing light energy transfer toan analyte, e.g., to ionize and desorb it from the surface of thesupporting substrate. The metal may be selected from the groupcomprising transition metals including Fe, Co, Cr, Ni, Mo, W, V, Cu, Znand precise metals including Ag, Au, Pt, Pd, Ru, Rh, or metal alloysthereof.

The active layer preferably has a thickness of between about 5 and 200nm, for example, between about 10 and 50 nm. When silicon is used as theactive layer, the silicon active layer is deposited on the thin filmlayer by a chemical vapor deposition process or by other thermalevaporation method. For example, it has been shown that depositing thesilicon active layer in a CVD oven at a temperature of about 480 degreesCelsius for a time period of between about 10 and 80 minutes, forexample, between about 30 and 80 minutes, provides the optimum massspectrometry results. For example, FIGS. 9A-B illustrate the differencein peak signal for mass spectrometry analysis of 25 picograms ofhaloperidol spotted onto a silicon coated nanostructured alumina surfacethat had silicon deposition times in the CVD oven of thirty minutes(FIG. 9A) and ten minutes (FIG. 9B). It will be appreciated that thehaloperidol peak only appears on the silicon coated surface that was CVDdeposited for about 30 minutes. Thus, the CVD deposition time (and hencesilicon active layer thickness) plays an important role in thesensitivity of the mass spectrometry signal.

The active layer can also optionally be coated or functionalized, e.g.,to enhance or add specific properties. For example, polymers, ceramics,or small molecules can optionally be used as coating materials. Theoptional coatings can impart characteristics such as water resistance,improved mechanical, optical (e.g., enhancement of light coupling) orelectrical properties or specificities for certain analytes. The activelayer may also be derivatized with one or more functional moieties(e.g., a chemically reactive group) to enhance laser desorption such asone or more silane groups, e.g., one or more per-fluorinated silanegroups, or other coatings such as diamond coatings, a hydrocarbonmolecule, a fluorocarbon molecule, or a short chain polymer of bothtypes of molecules which may be attached to the active layer via silanechemistry. Those of skill in the art will be familiar with numerousfunctionalizations and functionalization techniques which are optionallyused herein (e.g., similar to those used in construction of separationcolumns, bio-assays, etc.).

For example, details regarding relevant moiety and other chemistries, aswell as methods for construction/use of such, can be found, e.g., inHermanson Bioconjugate Techniques Academic Press (1996), Kirk-OthmerConcise Encyclopedia of Chemical Technology (1999) Fourth Edition byGrayson et al. (ed.) John Wiley & Sons, Inc., New York and inKirk-Othmer Encyclopedia of Chemical Technology Fourth Edition (1998 and2000) by Grayson et al. (ed.) Wiley Interscience (print edition)/ JohnWiley & Sons, Inc. (e-format). Further relevant information can be foundin CRC Handbook of Chemistry and Physics (2003) 83^(rd) edition by CRCPress. Details on conductive and other coatings, which can also beincorporated onto the active layer of the invention by plasma methodsand the like can be found in H. S. Nalwa (ed.), Handbook of OrganicConductive Molecules and Polymers, John Wiley & Sons 1997. See also,“ORGANIC SPECIES THAT FACILITATE CHARGE TRANSFER TO/FROM NANOCRYSTALS,”US Patent Publication No. US20040178390. Details regarding organicchemistry, relevant for, e.g., coupling of additional moieties to afunctionalized surface can be found, e.g., in Greene (1981) ProtectiveGroups in Organic Synthesis John Wiley and Sons, New York, as well as inSchmidt (1996) Organic Chemistry Mosby, St Louis, Mo., and March'sAdvanced Organic Chemistry Reactions, Mechanisms and Structure, FifthEdition (2000) Smith and March, Wiley Interscience New York ISBN0-471-58589-0,and US Patent Publication No. US20050181195, entitled“Super-hydrophobic Surfaces, Methods of Their Construction and UsesTherefor.” Those of skill in the art will be familiar with many otherrelated references and techniques amenable for functionalization ofsurfaces herein. The analytes may be directly linked to the active layersurface, e.g., through silane groups, or may be coupled via linkerbinding groups or other appropriate chemical reactive groups toparticipate in linkage chemistries (derivitization) with linking agentssuch as, e.g., substituted silanes, diacetylenes, acrylates,acrylamides, vinyl, styryls, silicon oxide, boron oxide, phosphorusoxide, N-(3-aminopropyl)3-mercapto-benzamide,3-aminopropyl-trimethoxysilane, 3-mercaptopropyl-trimethoxysilane,3-maleimidopropyl-trimethoxysilane, 3-hydrazidopropyl-trimethoxysilane,trichloro-perfluoro octyl silane, hydroxysuccinimides, maleimides,haloacetyls, pyridyl disulfides, hydrazines, ethyldiethylaminopropylcarbodiimide, and/or the like.

The supporting substrate 10 may be made from any one of a number ofconventional materials including stainless steel, glass, quartz,semiconductor materials such as a semiconductor wafer, silicon,polymers, ceramics etc. Samples of the substances (e.g., smallmolecules, proteins, peptides etc.) to be analyzed are optionally placedin contact with the active layer (e.g., directly or via the use or oneor more functional moieties such as one or more silane groups describedabove) by conventional dispensing means such as pipetting, dot-printingetc. Those of skill in the art will be familiar with various protocolsto follow to dry the samples for analysis. Laser energy levels and pulsedurations are also optionally optimized for analysis of the samplesarrayed upon the nanostructured substrate surface. Again, those of skillin the art will be familiar with ways of determining optimal parametersfor laser energy, pulse time, etc. for mass spectrometry. For example,the different parameters are optionally modified depending upon, e.g.,the specific molecules being detected. For example, the laser energyused can optionally be adjusted (e.g., higher laser energy levels forpeptides as opposed to small molecules). An interesting feature of thenanostructured surface of the present invention is that it requiresrelatively low laser energy (e.g., on the order of about 60 Hz) todesorb small molecules therefore reducing background ion interference.

In another aspect of the present invention, a mass spectrometry deviceis disclosed (e.g., for matrix-free analysis of small molecules such asproteins, peptides, or small molecule drugs) which comprises asupporting substrate having a first surface and a plurality ofnanocrystals, e.g., semiconductor nanocrystals such as quantum dots,deposited on the surface of the substrate. For example, the nanocrystalscan be a gold nanoparticle, a cobalt nanoparticle, an iron oxidenanoparticle, or a semiconductor nanocrystal including a semiconductormaterial, such as a Group IV compound (e.g., Si, Ge), a Group II-VIcompound, a Group II-V compound, a Group III-VI compound, a Group III-Vcompound, a Group IV-VI compound, a Group I-III-VI compound, a GroupII-IV-VI compound, or a Group II-IV-V compound. The semiconductormaterial can be, for example, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, HgTe, AlN, AIP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, GaSe, InN, InP,InAs, InSb, TIN, TIP, TIAs, TlSb, PbS, PbSe, PbTe, or mixtures thereof.The nanoparticle can be coated with one or more second materials such asa second metal, semimetal, polymer, or semiconductor material such asZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgO, MgS, MgSe, MgTe, HgO,HgS, HgSe, HgTe, AIN, AIP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP,InAs, InSb, TIN, TIP, TIAs, TlSb, TlSb, PbS, PbSe, PbTe, or mixturesthereof.

In one embodiment, the nanoparticles are spin-coated onto the substratesurface, and then plasma treated to generate a water adsorbable oxidesurface. The nanoparticles can also be spotted from solution onto thesubstrate surface (e.g., alone or in combination with the molecules tobe sampled such as low molecular weight proteins, peptides or smallmolecule drugs), or can be applied to the surface by any other suitablemethod known to those of ordinary skill in the art. Alternatively, thenanocrystals can be applied directly to a tissue section whereuponenergy transfer occurs from the nanocrystals directly to molecules inthe tissue sample (such as proteins or small molecule drugs) which wouldbe desorbed and ionized for mass spectrometry analysis. The surface ofthe nanoparticles can be functionalized as necessary. In certainembodiments, the collection or population of nanocrystals issubstantially monodisperse in size and/or shape. See, e.g., U.S. Pat.No. 6,207,229 (Mar. 27, 2001) and U.S. Pat. No. 6,322,901 (Nov. 27,2001) to Bawendi et al. entitled “Highly luminescent color-selectivematerials,” the entire contents of each of which are incorporated byreference herein.

While any method known to the ordinarily skilled artisan can be used tocreate nanocrystals suitable for use in the present invention, suitably,a solution-phase colloidal method for controlled growth of inorganicnanocrystals is used. See Alivisatos, A. P., “Semiconductor clusters,nanocrystals, and quantum dots,” Science 271:933 (1996); X. Peng, M.Schlamp, A. Kadavanich, A. P. Alivisatos, “Epitaxial growth of highlyluminescent CdSe/CdS Core/Shell nanocrystals with photostability andelectronic accessibility,” J. Am. Chem. Soc. 30:7019-7029 (1997); and C.B. Murray, D. J. Norris, M. G. Bawendi, “Synthesis and characterizationof nearly monodisperse CdE (E=sulfur, selenium, tellurium) semiconductornanocrystallites,” J. Am. Chem. Soc. 115:8706 (1993). This manufacturingprocess technology leverages low cost processability without the needfor clean rooms and expensive manufacturing equipment. In these methods,metal precursors that undergo pyrolysis at high temperature are rapidlyinjected into a hot solution of organic surfactant molecules. Theseprecursors break apart at elevated temperatures and react to nucleatenanocrystals. After this initial nucleation phase, a growth phase beginsby the addition of monomers to the growing crystal. The result isfreestanding crystalline nanoparticles in solution that have an organicsurfactant molecule coating their surface.

Utilizing this approach, synthesis occurs as an initial nucleation eventthat takes place over seconds, followed by crystal growth at elevatedtemperature for several minutes. Parameters such as the temperature,types of surfactants present, precursor materials, and ratios ofsurfactants to monomers can be modified so as to change the nature andprogress of the reaction. The temperature controls the structural phaseof the nucleation event, rate of decomposition of precursors, and rateof growth. The organic surfactant molecules mediate both solubility andcontrol of the nanocrystal shape. The ratio of surfactants to monomer,surfactants to each other, monomers to each other, and the individualconcentrations of monomers strongly influence the kinetics of growth. Insuitable embodiments, Si or CdSe is used as the nanocrystal material,although other suitable semiconductor materials can be used as describedabove (e.g., InP, ZnS, and the like).

By controlling the size and composition of the semiconductornanocrystals used in the practice of this embodiment of the presentinvention, the nanocrystals will absorb laser light of a particularwavelength, or a particular range of wavelengths, whereupon efficientenergy transfer from the nanocrystal to the molecules deposited thereonor therewith can occur to desorb and ionize the molecules for massspectrometry analysis. The ability to make nanocrystals out of differentsemiconductors, and to control their size and shape, allows for thefabrication of nanocrystals that will absorb light from the UV, tovisible, to near infrared (NIR), to infrared (IR) wavelengths.Nanocrystals for use in the present invention will suitably be less thanabout 100 nm in size, and down to less than about 2 nm in size. Insuitable embodiments, the nanocrystals of the present invention absorbultraviolet light, e.g., at a wavelength of between about 300 and 400nm.

In another aspect of the invention, a device for enhanced surface areaapplications and/or hydrophobic surface applications is disclosed whichgenerally comprises a supporting substrate, a non-silicon film layer onat least a region of the surface, the film layer having a nanostructuredsurface, and a hydrophobic (or hydrophilic) coating deposited on thefilm layer. The hydrophobic coating may comprise, for example, adiamond-like carbon coating (e.g., an amorphous diamond film) or othercoating which renders the surface of the device hydrophobic (orhydrophilic). The diamond-like carbon film can be applied to thenanostructured surface by, e.g., using pulsed laser ablation on agraphite target or by a plasma assisted CVD method at low temperature.When the deposited material is heated, such films lose their stress, yetretain their diamond-like properties.

Such devices can be used in a variety of high surface area and/orhydrophobic or hydrophilic surface applications including those whichare disclosed in greater detail in co-pending and related cases U.S.Pat. No. 7,074,294, which is a continuation-in-part of U.S. Pat. No.7,056,409, which claims priority to U.S. Provisional Patent ApplicationNo. 60/463,766, filed Apr. 17, 2003; and U.S. patent application Ser.No. 10/833,944, filed Apr. 27, 2004, which claims priority to U.S.Provisional Application Ser. No. 60/466,229, filed Apr. 28, 2003; and toU.S. patent application Ser. No. 10/840,794 filed May 5, 2004, which isa continuation-in-part of U.S. patent application Ser. No. 10/792,402,filed Mar. 2, 2004, which claims priority to U.S. Provisional PatentApplication Ser. Nos. 60/468,390, filed May 6, 2003 and 60/468,606 filedMay 5, 2003, each of which has previously been incorporated by referencein their entirety herein.

For example, as disclosed in the above-referenced applications, theunique nanostructured surfaces disclosed herein can be used in, on orwithin various medical devices, such as clamps, valves, intracorporealor extracorporeal devices (e.g., catheters), temporary or permanentimplants, stents, vascular grafts, anastomotic devices, aneurysm repairdevices, embolic devices, implantable devices (e.g., orthopedicimplants) and the like. Such enhanced surfaces provide many enhancedattributes to the medical devices in, on, or within which they are usedincluding, e.g., to prevent/reduce bio-fouling, increase fluid flow dueto hydrophobicity, increase adhesion, biointegration, etc. Suchnanostructured coatings can be used as surface coatings for touchscreens such as for information kiosks, gaming/entertainment/mediaconsoles, point-of-sale terminals, industrial and medicalinstrumentation, ATM machines, kiosks in retailing, personal computermonitor screens, automobile displays, and the like. The nanostructuredfilms disclosed herein can be used to provide a surface for cellattachment, differentiation, and proliferation, as a substrate topromote cell growth, or as a substrate for DNA or protein microarrays,e.g., to hybridize nucleic acids, proteins and the like. Thenanostructured films disclosed herein have applications in vivo fortissue grafting including osteoblasts, neuronal, glia, epidermal,fibroblast cells and the like. Such nanostructured coatings can also becombined in particular applications with other nanostructured componentssuch as nanofibers or nanowires which can be grown on the surfacesand/or deposited thereon, to provide further attributes of increasedadhesion, hydrophobicity, hydropholicity, conductivity (e.g., forelectrical contact applications) and the like to the devices with whichthey are used.

EXAMPLES Example 1

The following non-limiting example demonstrates the feasibility of usingan alumina nanostructured underlying thin film coated with an activesilicon layer to perform mass spectrometry analysis. The nanostructuredsurface used in these experiments is based on alumina. 50 nm of aluminawas sputtered onto a 40 mm square polished stainless steel plate. Theplate was exposed to boiling water for three minutes which converts thealumina into a highly structured surface.

The plate was then oxygen plasma cleaned for 10 minutes before placingit into a CVD furnace where silicon was deposited on it for 70 minutes.Two steel plates without the nanostructured alumina were also placedinto the furnace as controls, one with no coating (FIG. 5A) and theother with a silicon film layer deposited thereon but no underlyingalumina nanostructured surface (FIG. 5B). When the plates were removedfrom the furnace they were exposed to trichloro-perfluoro octyl silane(Gelest, Morrissville, Pa.) vapor for four hours to render themhydrophobic along with a plane steel plate. The plates were spotted with0.5 ul of Prazosin in a 50% acetonitrile water mix, allowed to dry andthen inserted into an ABI Voyager DE-pro mass spectrometer (availablecommercially from Applied Biosystems, Foster City, Calif.) for analysis.

FIG. 5A shows results of mass spectrometry analysis of samples on thestainless steel substrate without any nanostructured thin film layer orsilicon film layer. FIG. 5B shows results of mass spectrometry analysisof samples on a stainless steel substrate with a silicon layer andwithout any underlying nanostructured thin film layer. FIG. 5C showsresults of mass spectrometry analysis of samples on a stainless steelsubstrate with a thin nanostructured film layer and a silicon activelayer according to the teachings of the present invention. As can beseen almost no signal can be detected on the steel or silicon treatedsurfaces, but with the underlying alumina nanostructured surface, thePrazosin peak is off scale representing more then a 100× improvement.

FIGS. 6A-D show further examples of mass spectrometry analysis of 25picograms of four pharmaceutical compounds (e.g., clonidine,haloperidol, prazosin, and quinidine, respectively) deposited on asilicon coated nanostructured alumina surface. FIGS. 7A-D show examplesof mass spectrometry analysis of 2.5 picograms of those same fourcompounds deposited on a silicon coated nanostuctured alumina surface.As can be appreciated, the mass spectra demonstrate signal detection ofall four molecules spotted at 25 pg and 2.5 pg, respectively.

Example 2

The following non-limiting example demonstrates the feasibility of usingan aluminum nanostructured underlying thin film coated with an activetungsten layer to perform mass spectrometry analysis. The nanostructuredsurface used in these experiments is based on aluminum. 100 nm ofaluminum was sputtered onto a 40 mm square polished stainless steelplate. The plate was exposed to boiling water for five minutes whichconverts the aluminum into a highly structured surface.

The plate was then oxygen plasma cleaned for 10 minutes before placingit into a high vacuum sputtering system (UHV Sputtering Inc., San Jose,Calif.) where tungsten was deposited on it to a depth of 50 nm. Theplate was then plasma cleaned again and was exposed totrichloro-perfluoro octyl silane (Gelest, Morrisville, Pa.) vapor forthree hours to render it hydrophobic. The plate was then washedthoroughly in methanol and baked for 20 minutes at 100 degrees Celsius.The plate was spotted with 25 pg of prazosin, haloperidol, quinidine andclonidine in a 50% acetonitrile water mix, allowed to dry and theninserted into an ABI Voyager DE-pro mass spectrometer (availablecommercially from Applied Biosystems, Foster City, Calif.) for analysis.

FIGS. 10A-D show results of mass spectrometry analysis of 25 picogramsof the four pharmaceutical compounds (e.g., prazosin, haloperidol,quinidine and clonidine, respectively) deposited on the tungsten coatednanostructured aluminum surface. As can be appreciated, the mass spectrademonstrate signal detection of all four molecules spotted at 25 pg.

While the foregoing is directed to preferred embodiments of the presentinvention, other and further embodiments of the invention may be devisedwithout departing from the basic scope thereof, and the scope thereof isdetermined by the claims which follow.

1. A method for manufacturing a device useful for analysis of one ormore analytes comprising: providing a supporting substrate having afirst surface and a first film layer deposited on the first surface;forming a nanostructured surface from the first film layer by (a)exposing the film layer to water or water vapor at a temperature ofbetween about 90 to 100 degrees Celsius or (b) autoclaving the filmlayer; depositing a second layer comprising silicon or tungsten on thenanostructured surface; and depositing one or more analytes onto one ormore regions of the second layer.
 2. The method of claim 1, furtherirradiating the one or more analytes with a laser to ionize and desorbthe analytes from the one or more regions of the second layer.
 3. Themethod of claim 1, wherein the first film layer comprises alumina oraluminum.
 4. The method of claim 3, wherein the second layer comprisessilicon.
 5. The method of claim 4, wherein the second layer is depositedon the first film layer by a chemical vapor deposition process.
 6. Themethod of claim 5, wherein the step of forming a nanostructured surfacecomprises exposing the alumina or aluminum film to water or water vaporat a temperature of between about 90 to 100 degrees Celsius for betweenabout five to thirty minutes.
 7. The method of claim 6, furthercomprising functionalizing the surface of the second layer with one ormore functional groups.
 8. The method of claim 7, wherein the one ormore functional groups comprise a functional organic layer comprising ahydrocarbon molecule, a fluorocarbon molecule, or a short chain polymerof both types of molecules.
 9. The method of claim 8, wherein the one ormore functional groups comprise a functional organic layer comprising afluorocarbon molecule.
 10. The method of claim 1, wherein said firstfilm layer has a thickness of between about 10 nm to 1000 nm.
 11. Themethod of claim 10, wherein said first film layer has a thickness ofbetween about 100 nm and 200 nm.
 12. The method of claim 1, wherein saidsecond layer has a thickness between about 5 and 200 nm.
 13. The methodof claim 12, wherein said second layer has a thickness between about 10and 50 nm.